MD simulations of wild type and mutated E.coli LeuRS CP1 domain complexed with pre-transfer editi

ng substrate analog

Haimei Zhu

June 12th, 2008

Aminoacyl-tRNA synthetases (aaRS) • aaRS are responsible for ac

curate matching each amino acid with its cognate tRNA

• Some synthetases have editing activities that clear the wrong amino acids

Homology model of E.coli LeuRSAmy M. Williams. et al PNAS. 2006, 103, 3586-3591

Leucyl-tRNA synthetases (LeuRS)

Mis-Aminoacylation of 4µM tRNALeu with isoleucine by 1µM E.coli LeuRSs

• E.coli LeuRS relies only on post-transfer editing

• Mutation T252Y prevents post-transfer editing activity

• Double mutation T252Y/A293D rescues pre-transfer editing activity Amy M. Williams. et al PNAS.

2006, 103, 3586-3591

Isoleucyl-adenylate NvaAMSTommie L. Lincecum, Jr. et al Molecular Cell. 2003, 11, 951-963

PDB 1BOH

T . Thermophilus LeuRS

Work plan

To study the rescued pre-transfer editing activity• MD simulation Systems of E. coli LeuRS CP1

domain complexed with pre-transfer substrate analog NvaAMS:

– Wild type – Single mutant T252Y – Single mutant A293D– Double mutant T252/A293D

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LeuRS CP1

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Residues

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0.5LeuRS CP1 T252Y

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0.5LeuRS CP1 A293D

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LeuRS CP1 T252Y/A293D

Two large flexible regions: residues 280-300 and 360-390

The region in red circle form the whole active site pocket for NvaAMS and regions adjacent to region 360-390 are quite stable

It is reasonable to consider only circled region (residues 228-352) for complex interaction studies

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LeuRS CP1 A293D

Black line: Backbone of Protein, Red line: Backbone of residues 228-352, Green line: NvaAMS

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0.4 LeuRS CP1 T252Y/A293D

Proposed hydrolysis mechanism

• No proven directly catalytic residue • Anhydride and ester linkages in pre- and post substrate

hydrolyzed by water attacking the carbonyl carbon• Nucleophilic OH- • Positive charge of the carbonyl carbon• Favorable substrate binding configuration• Active site residues positioned water molecule

NvaAMSIsoleucyl-adenylate

Wild type system

• Water analysis– Extract water molecules within 0.35 nm of carbon

yl carbon from trajectories– Find the loyal water molecules

• Hydrogen bond analysis– Extract active site residues which form hydrogen

bond to carbonyl oxygen from trajectories

• Clustering– based on the conformational states of NvaAMS

Most popular NvaAMS conformation and loyal waters in simulation 1

Cluster occupancy 36%

water occupancy 31%

Carbonyl oxygen –THR228OG hydrogen bond occupancy 65% , occurs in the last 6 ns

Most popular NvaAMS conformation and loyal waters in simulation 2

Cluster occupancy 56%

water occupancy 78%

Carbonyl oxygen –THR228OG hydrogen bond occupancy 0.3%

Most popular NvaAMS conformation and loyal waters in simulation 3

Cluster occupancy 38%

water occupancy 22%

Carbonyl oxygen –THR228OG hydrogen bond occupancy 30%, occurs in the last 3 ns

Future plan

• Water analysis and hydrogen bond analysis for other systems